The goal of this work is to understand the molecular mechanisms that govern the assembly of proteins into an organized cellular domain. The experiments will examine domain assembly in bacterial cells, but the establishment of domains is a general phenomenon that occurs in all cell types. Given this project as proof- of-principle, the same experimental approach could be transferred to eukaryotic systems and used to make new discoveries in human biology. The work will focus on cell pole assembly in the rod shaped Caulobacter crescentus, which creates two functionally distinct poles at opposite ends of a contiguous cytoplasm. These poles are complex domains that carry out multiple functions, including flagellar assembly, chromosome positioning, and the regulation of transcription factor activity. Polar assembly factors will be discovered in two genetic screens: one a visual screen to directly identify genes that are required for the proper localization of known polar proteins, the other a bioinformatic screen for new polar proteins. For every gene identified in the screens, upstream and downstream factors in the assembly process will be identified using genetic, microscopic, and biochemical methods. The bioinformatic screen has already yielded a novel protein that seems to use chromosome replication as a cue for assembly at the cell pole. A key aspect of the analysis will be a precise determination of protein localization using protein-specific labeling techniques for cryo-EM tomography. This will reveal the arrangement of proteins and functional complexes in the context of domain ultrastructure, thereby characterizing an aspect of assembly that is inaccessible by genetic and biochemical methods. The project will define a temporal order of assembly steps and show how individual components are placed in the overall architecture of the cell pole, providing a detailed four dimensional view of the construction of this domain. Proteins are made as individual subunits, and must find a way to fit together with other proteins to create larger complexes with biological function. Such assembly must occur properly in order for our cells to grow and divide, and the flip side of the coin is that we may block this process in viruses and bacteria to inhibit their growth. I am studying protein assembly in a bacterium, which could lead directly to the development of new antibiotics. The work will also have broader impact on our knowledge of how complex assembly contributes to cellular function in all life forms. [unreadable] [unreadable] [unreadable]